MS sample preparation by centrifugation using OMIX C18 tips_Gel plugs
Day 1
Cut gel plugs, chop each one into 1mm3 cubes (about 25 pieces per gel plug)
Add 500ul 50mM AmBic /50% ACN
Mix with the gel pieces by vortexing
If the color of the solution changes to dark blue immediately, discard it and add another 500ul 50mM AmBic /50% ACN (Can use 1ml here or use 500ul each time and repeat multiple times)
Incubate @ 37°C for 1hr; discard the sup
Add another 500ul 50mM AmBic /50% ACN if the gel pieces are still blue
Destain as needed (overnight okay)
Remove 50mM AmBic /50% ACN when the gel pieces are completely destained
Add 500ul ACN, vortex, let the tubes set at RT for 2’ or more
Remove ACN from all samples
Dehydrate in speed vac for 10’
Samples can be stored @ -20°C or proceed to trypsin digest
Day 2
Prepare trypsin working solution: 12.5ng/ul in 50mM AmBic (trypsin stock 100ng/ul in 1mM HCl)
Prepare tubes on ice
Add 12.5ng/ul trypsin to cover (38ul per tube for 15-well gel plugs or 90ul for 10-well gel plugs; gels are 1mm thick)
Let samples swell for 45’ on ice
After swelling, add 50mM Ambic to cover gel pieces if necessary (~12ul for 15-well gel or 30ul for 10-well gel)
Move the tubes to 37°C incubator to digest overnight (6 hrs at least) Day 3 (The gel extraction steps here are different from what Kelly does)
Add 0.5% TFA (w/v, final concentration) to stop digestion • Dilute 20% TFA stock to 2% first • Add 1/3 of digestion vol. of 2% TFA to digestion mix • Mix FTA with gel pieces and incubate @ RT for 5’
Remove the digest solution to 0.65ml low-binding tubes; hold @ RT or 4°C
Extract with 50ul of 0.1% TFA @ RT for 45’
Remove extractant, combine with digest solution (total vol. will be ~100ul for each 15-well gel plug)
Desalt sample using OMIX tips (C18, 100ul for gel plugs) by centrifugation
Tip wet: 2x 100ul of 50% ACN (v/v), 0.1% TFA (w/v)
Load the solution in OMIX tip on top of the filter; Try spin @ 500rpm for 1’ first to see how it goes, increase time or speed only if liquid passes through too slowly; Do the same thing for later spinning steps, start slow and short time to avoid over-dry of the tip (500rpm for 1’ is sufficient for all spinning steps)
Spin @ 500rpm for 1’
Tip wash: 2x 100ul of 0.1% TFA (w/v)
Sample binding: let the sample passes through the filter once
Save the flow-through @ -20°C in case the binding is not complete
Sample wash: 4x 100ul of 0.1% TFA (w/v) This is the last wash step before elution; spin harder at the last wash to let all liquid pass through
Elute sample by centrifugation
Pre-wash one set of low-binding tubes to hold elution (both E1 and E2) with 100% ACN
Elution 1: 80ul 40%ACN / 0.5% HAc Use clean collecting tube (pre-washed with 100% ACN) Let everything pass through
Elution 2: 80ul 80%ACN / 0.5% HAc Let everything pass through Collect E1 and E2 in the same tube
Put the tube containing E1+E2 in the speed vac and dry completely